This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT ₁ )-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT ₁ -receptor antagonist–stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT ₁ -receptor antagonists on platelet adhesion to collagen and thromboxane A ₂ analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 μmol/L, which was attenuated by NO synthase inhibitor N ^G -nitro- l -arginine methyl ester. The angiotensin II AT ₁ -receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT ₁ -receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N ^G -nitro- l -arginine methyl ester. Neither the AT ₂ receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A ₂ /prostaglandin H ₂ receptor antagonist SQ29,548 had any effect on angiotensin II AT ₁ -receptor antagonist–stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT ₁ -receptor antagonists.

Summary: There is an increased number of in vitro evidence that angioten-sin II (Ang II) may promote thrombosis. However there are no in vivo experiments exploring the effect of Ang II on thrombus formation. In the present study we have investigated the influence of Ang II on venous thrombosis in renovascular hypertensive rats. Furthermore, we examined the role of AT1 receptor and Ang II metabolites: angiotensin III (Ang III) and angioten-sin IV (Ang IV) in the mechanisms of Ang II action. The contribution of coagulation and fibrinolytic systems in the mode of Ang II action was also determined. Venous thrombosis was induced by ligation of vena cava. Ang II infused into rats developing venous thrombosis caused dose-dependent increase in thrombus weight, which was partially reversed by losartan, selective AT1 antagonist. Ang III did not influence the thrombus formation in hypertensive rats, while Ang IV caused a marked increase in thrombus weight only in one of the used doses. Our study shows that Ang II via AT1 receptor enhances thrombosis development. The prothrombotic effect of Ang II may partially depend on enhanced leukocytes adhesion to endothelial cells accompanied by accelerated fibrin formation and increased plasma level of PAI-1. Moreover, Ang II action is partially mediated by one of its metabolites – Ang IV.

Introduction: We investigated the role of primary haemostasis, fibrinolysis, nitric oxide (NO) and oxidative stress as well as mineralocorticoid receptors (MR) in acute aldosterone prothrombotic action. Materials and methods: Venous thrombosis was induced by stasis in Wistar rats. Aldosterone (ALDO; 10, 30, 100 µg/kg/h) was infused for 1 h. Eplerenone (EPL; 100 mg/kg, p.o.), a selective MR antagonist, was administered before ALDO infusion. Bleeding time (BT) and platelet adhesion to collagen were evaluated. The expression of nitric oxide synthase (NOS), NADPH oxidase, superoxide dismutase (SOD) and plasminogen activator inhibitor (PAI-1) was measured. NO, malonyl dialdehyde (MDA) and hydrogen peroxide (H₂O₂) plasma levels were assayed. Results: Significant enhancement of venous thrombosis was observed after ALDO infusion. ALDO shortened BT and increased platelet adhesion. Marked increases were observed in PAI-1, NADPH oxidase and SOD mRNA levels. MDA and H₂O₂ levels were augmented in ALDO-treated groups, and NOS expression and NO level were decreased. EPL reduced ALDO effects on thrombus formation, primary haemostasis, PAI-1 expression and MDA level. Conclusion: Short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, fibrinolysis, NO and oxidative stress-dependent pathways. The MR antagonist only partially diminished the ALDO effects, suggesting the involvement of additional mechanisms.

Angiotensin-(1-7) [Ang-(1-7)] is the bioactive peptide which may be responsible for some of the pharmacological effects of losartan. Our previous study has demonstrated the antithrombotic action of losartan in a model of experimental thrombosis. In the present study, we compared the antithrombotic action of losartan and Ang-(1-7). Acute (10 mg/kg, p.o.) and chronic (10 mg/kg, p.o., three weeks) losartan administration to spontaneously hypertensive rats (SHR) induced a decrease in thrombus weight (1.6±0.6 mg and 1.2±0.3 mg respectively vs. control 2.9±0.8 mg; p<0.05, p<0.05). A similar reduction was observed in two-kidney, one-clip hypertensive rats (2K-1C) receiving acute losartan administration (1.39±0.29 mg vs. 3.25±0.62 mg; p<0.01). Infusion of Ang-(1-7) to 2K-1C rats also reduced the thrombus weight (1.01±0.34 mg, 1.23±0.38 mg and 2.17±0.36 mg for 1, 10, 100 pmol/kg/min, respectively vs. 3.58±0.6 mg control; p<0.01, p<0.01, p<0.05). Losartan produced a decrease in systolic blood pressure (BP) in SHR as well as in 2K-1C rats, while Ang-(1-7) infusion had no effect on BP. Acute losartan dosing to 2K-1C rats decreased platelet adhesion to fibrillar collagen (24.9±1.0% vs. control 31.5±1.1%, p<0.001). The incubation of platelet samples with Ang-(1-7) (10^-6 and 10 ^—5 M) also reduced adhesion to fibrillar collagen (38.4±0.1% and 33.8±0.8% respectively vs. control 40.0±0.6%; p<0.05, p<0.001). There were no apparent changes in prothrombin time, activated partial thromboplastin time and euglobulin clot lysis time in losartan and Ang-(1-7)-treated groups. We conclude that, like losartan, Ang-(1-7) is able to act as an antithrombotic agent.